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United States Department of Agriculture

Agricultural Research Service

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Research Project: Identification, Characterization, and Validation of Genetic Mutations Incurred During in Vitro Attenuation

Location: Avian Disease and Oncology Laboratory

2010 Annual Report


1a.Objectives (from AD-416)
Marek’s disease virus (MDV), unlike most other viruses, is highly cell-associated and consequently, exists as population rather due to the inherent process of mutations. This makes it very difficult to determine the relationship between genetic variation and phenotypic variation. With the advent of molecular clones of MDV-BAC clones and genomic technologies, we can now better address how specific genetic changes influence virus virulence, vaccine efficacy, etc. Specifically, we would like to know during cell passage, are there preferred sites that are required for MDV attenuation. The proposed experiments will guide scientists on how to molecular characterize MDV field strains, what changes occur during cell passage attenuation and vaccine production, and other fundamental knowledge.


1b.Approach (from AD-416)
Our virulent BAC cloned MDV genome that generates a fully virulent virus will be passed in cell culture, which is known to attenuate the virus. At every 10 passages, the viral population will be used to challenge chickens to determine the amount of MD incidence. This will continue until the viral population is completely avirulent. Preceeding populations will have their viral genome purified and sequenced using next generation sequencers (e.g., Illumina GA or ABI SOLiD) to identify polymorphisms in the genome as well as the allele frequency. In addition, RNAs from the same sequenced populations will be sequenced, which when combined with the genomic sequence information, will confirm polymorphisms and reveal changes in viral gene transcription pattern. Following analysis, key genetic changes will be introduced into the virulent viral genome to address whether the polymorphisms do promote attenuation.


3.Progress Report

This project is linked to Specific Cooperative Agreement 3635-32000-015-12S "Identification, Characterization, and Validation of Genetic Mutations Incurred During In Vitro Attenuation of Marek’s Disease Virus." Marek's disease (MD) is one of the most serious chronic threats to the U.S. poultry industry due to recurring yet unpredictable vaccine breaks. Genetic mutations in Marek's disease virus (MDV), the causal pathogen, play a pivotal role in both the evolution of field strains with higher virulence and in vitro (cell culture) attenuation, the process used to make traditional vaccines. Unfortunately, little is known about naturally-occurring MDV mutations and their influences on virulence. This year, starting with our infectious BAC (plasmid) clone containing the entire MDV genome, we generated the virus and passed triplicate replications in chicken cells. At cell passage 60 and 70, all three replicates became completely attenuated based on challenge experiments in highly susceptible chickens. This indicates and confirms past results to show that the viral genome picks up random mutations that will eventually attenuate the virus. These attenuated populations form the basis for future studies to identify all the changes in the viral genome. This information will help promote the rational design of superior MD vaccines, provide molecular markers of MDV pathotypes, and aid in the prediction of MDV evolution in the field. This project is monitored by monthly e-mail and telephone calls between the two parties and, when possible, direct interactions at scientific meetings.


Last Modified: 9/23/2014
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